MOTIVATION: G-quadruplexes (G4) are important regulatory non-B DNA structures with therapeutic potential. A tool for rational design of mutations leading to decreased propensity for G4 formation should be useful in studying G4 functions. Although tools exist for G4 prediction, no easily accessible tool for the rational design of G4 mutations has been available. RESULTS: We developed a web-based tool termed G4Killer that is based on the G4Hunter algorithm. This new tool is a platform-independent and user-friendly application to design mutations crippling G4 propensity in a parsimonious way (i.e., keeping the primary sequence as close as possible to the original one). The tool is integrated into our DNA analyzer server and allows for generating mutated DNA sequences having the desired lowered G4Hunter score with minimal mutation steps. AVAILABILITY AND IMPLEMENTATION: The G4Killer web tool can be accessed at: http://bioinformatics.ibp.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• MeSH
• Algorithms MeSH
• DNA MeSH
• G-Quadruplexes * MeSH
• Mutation MeSH
• Sequence Analysis, DNA MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1-10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.

• MeSH
• Benzothiazoles metabolism   MeSH
• Fluorescence MeSH
• G-Quadruplexes MeSH
• Genetic Variation * MeSH
• Templates, Genetic * MeSH
• Reproducibility of Results MeSH
• RNA genetics   MeSH
• Saccharomycetales genetics   MeSH
• Base Sequence MeSH
• Telomerase genetics   MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

• MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA metabolism   MeSH
• G-Quadruplexes MeSH
• Telomere Homeostasis physiology   MeSH
• DNA, Single-Stranded metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides genetics   MeSH
• Telomere-Binding Proteins metabolism   MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Telomerase genetics   MeSH
• Telomere metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of 'genetic camouflage' to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material.

• MeSH
• Biological Evolution MeSH
• G-Quadruplexes * MeSH
• Genome, Human MeSH
• Genomics MeSH
• Humans MeSH
• Paleontology MeSH
• Hepatitis B virus * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Telomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.

• MeSH
• DNA Helicases genetics   metabolism   MeSH
• Genetic Loci MeSH
• Bryopsida enzymology   genetics   MeSH
• Mutation MeSH
• Genomic Instability * MeSH
• Rad51 Recombinase genetics   metabolism   MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 18S genetics   MeSH
• RNA, Ribosomal, 5S genetics   MeSH
• RNA, Ribosomal genetics   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Telomere genetics   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH